PROJECT SUMMARY Hepatitis C virus (HCV) is an important human pathogen that can cause severe liver diseases including acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Studies in recent years indicated that HCV could induce autophagy both in vitro and in vivo to enhance its replication. As autophagy plays an important role in maintaining cellular homeostasis, the prolonged perturbation of this pathway by HCV during chronic infection can have profound consequences on the progression of liver diseases in HCV- infected patients. Although significant progresses have been made during the past few years to understand the relationship between HCV and autophagy, many questions remain unanswered. The goal of this research is to continue our previous studies to further understand the relationship between HCV and autophagy. In Aim 1, we will continue our previous studies to investigate the biogenesis pathway of autophagosomes in HCV-infected cells by identifying the origin of these membrane vesicles and determine whether they are derived from the homotypic fusion of phagophores. As our recent studies indicated that HCV induced autophagy via a non-canonical pathway, in Aim 2, we will continue to delineate this pathway and examine the roles of HCV nonstructural proteins in the induction of this pathway. In addition, as our preliminary studies revealed the specific association of lipid rafts with autophagosomes induced by HCV, we will also investigate how lipid rafts are recruited to HCV-induced autophagosomes and their possible role in mediating HCV RNA replication on autophagosomal membranes. We have recently developed a novel approach to purify autophagosomes from HCV-infected cells for the identification of their associated protein factors. In Aim 3, we will continue to characterize these protein factors, which include annexin A2, apolipoprotein E and syntaxin 7, and determine their possible roles in the biogenesis of autophagosomes and HCV replication. Our proposed research will provide important information for understanding the interaction between HCV and its host cells and lead to a better understanding of the HCV life cycle and its pathogenesis. It will also provide important information for understanding the cellular autophagic pathway, which is frequently exploited by viruses to enhance their replication.